WO2003004508A1

WO2003004508A1 - Process for production of saccharide oxazoline derivatives

Info

Publication number: WO2003004508A1
Application number: PCT/JP2002/006575
Authority: WO
Inventors: Shin-Ichiro Shoda; Masaya Fujita; Masako Suenaga; Kenji Saito
Original assignee: Seikagaku Corporation
Priority date: 2001-07-02
Filing date: 2002-06-28
Publication date: 2003-01-16
Also published as: JP2003012683A; JP4866515B2; EP1405857A4; US20040176588A1; EP1405857A1; EP1405857B1

Abstract

A process for production of saccharide oxazoline derivatives represented by the general formula (2), characterized by reacting an acylamino sugar represented by the general formula (1) with a metal fluoride: (1) (2) wherein X is F, Cl, Br, or I; R1 is H or (CH2)n-CH3 (wherein n is 0 to 5); and R2, R3, R4, R5 and R6 are each independently H, N3, protected hydroxyl, protected amino, or Y-R7 (wherein Y is O, NH, or S; and R7 is a mono- or oligo-saccharide residue, with the proviso that when the residue bears OH, NH2 or COOH, the group(s) is protected), provided that at least one of R2 and R3 and at least one of R4 and R5 are each hydrogen.

Description

Description Method for producing sugar oxazoline derivative

The present invention relates to a method for producing a sugar oxazoline derivative. Background art

In recent years, sugar oxazoline derivatives have attracted attention as substrates for sugar chain synthesis using sugar chain-related enzymes, and methods for obtaining sugar oxazoline derivatives are being studied. Conventionally, tetraethylammonium chloride was used as a nucleophile and sodium bicarbonate was used as an acid scavenger in an acetonitrile solution of N-acetyl-3,4,6-tri-0-acetyl-hydarcosaminyl chloride. In addition, a method of producing a bicyclic sugar oxazoline derivative by reacting is used (JP-A-9-30888). However, in this method, it is difficult to remove the excess reaction reagent remaining in the reaction-finished solution, and a complicated purification technique is required to obtain a high-purity glycosoxazoline derivative. Disclosure of the invention

In recent years, the effects of oligosaccharides and glycosaminoglycans, which are biological substances, have been attracting attention in the medical field, and a sugar oxazoline derivative that can be a substrate for enzyme synthesis of the oligosaccharides and glycosaminoglycans can be industrialized. There is a need for an inexpensive and simpler manufacturing method.

The present inventors have conducted intensive studies to solve the above-mentioned problems, and as a result, by using metal fluoride as a reaction reagent, the fluoride exhibits both nucleophilicity and acid-capturing action, and the sugar oxazoline derivative It was found that the synthesis of the compound progressed, and that the removal of the fluoride was easy, and the present invention was completed.

The present invention provides a method for producing a sugar oxazoline derivative represented by the following general formula (2), which comprises reacting an acylamino sugar represented by the following general formula (1) with a metal fluoride. -

(Where X is selected from F, Cl, Br and I;

R ¹ is selected from H and (CH ₂ ) _n — CH ₃ (n = 0-5);

R \ R \ R \ R ⁵ and R ^6, which it independently, H, N _3, OH was protected by a protective group, NH ₂ protected by a protecting group and,, Y- R ⁷ (Y is 0 or NH or S, and R ⁷ is a residue of a monosaccharide or an oligosaccharide, and when OH, NH ₂ or CO OH is present, those groups are protected by a protecting group. Which are sugar residues). However, at least one of R ² and R ³ and at least one of R ⁴ and R ⁵ are H. )

(In the formula, R ¹ R ² , RRR ⁵ and R ⁶ are the same as described above.)

In addition, the present invention comprises reacting an acylamino sugar represented by the following general formula (1) with a metal fluoride, and then removing at least a part of the protecting group of the obtained sugar oxazoline derivative. This is a method for producing the sugar oxazoline derivative represented by the formula (3).

(Where X is selected from F, Cl, Br and I; R ¹ is selected from H and (CH ₂ ) _n — CH ₃ (n = 0-5);

R ² , R ³ , ⁴ ¹⁵ and ¹⁶ are each independently H, N ₃ , OH protected by a protecting group, NH ₂ protected by a protecting group, and Y—R ⁷ (Y is 0 or NH or S, and R ⁷ is a residue of a monosaccharide or an oligosaccharide, and when OH, NH ₂ or C〇OH is present, a monosaccharide or a group in which those groups are protected by a protecting group. Which are the residues of oligosaccharides). However, at least one of R ² and R ³ and at least one of R ⁴ and R ⁵ are H. )

(Wherein, R ¹ is as defined above,

^{^{R 2, R \ R 4,}} 11 5 and 1 ⁶ are each independently, H, N _3, which may be protected by a protective group OH, and good NH _2, be protected by a protecting group, Y—R ⁷ (Y is 0 or NH or S, and R ⁷ is a residue of a monosaccharide or an oligosaccharide, and when 〇H, NH ₂ or CO OH is present, those groups are protected by a protecting group. A monosaccharide or oligosaccharide residue which may be protected). However, at least one of R ² and R ³ and at least one of R ⁴ and R ⁵ are H. )

In both of the above production methods, the metal fluoride is preferably an alkali metal fluoride. BEST MODE FOR CARRYING OUT THE INVENTION

The method for producing the sugar oxazoline derivative represented by the general formula (2) of the present invention is characterized by reacting the acylamino sugar represented by the general formula (1) with a metal fluoride.

The acylamino sugar represented by the general formula (1) is not particularly limited depending on the origin and origin of the substance, and is necessary for those obtained from nature, those synthesized by genetic engineering using animal cells, microorganisms, etc. And the introduction of protecting groups in the usual way Can be used.

X can be selected from fluorine, chlorine, bromine and iodine classified as the same halogen, and chlorine is particularly preferable.

R ¹ can be selected from H and (CH ₂ ) _n —CH ₃ (n = 0 to 5), among which H, CH ₃ and CH ₂ CH ₃ are preferable, and CH ₃ is particularly preferable. These groups have little effect on the electronic state of the adjacent amide (one NHCO—).

^{^{^{R 2, R 3, R 4}}} , R 5 and R ⁶ are each defined above in H, N _3, OH was protected by a protective group, NH ₂ protected by a protecting group, YR ^Y (Y is 0 or NH ⁷ or S, and R ⁷ is a residue of a monosaccharide or an oligosaccharide, and when OH, NH ₂ or COOH is present, a group of the monosaccharide or the oligosaccharide whose group is protected by a protecting group. Residue). However, at least one of R ² and R ³ and at least one of R ⁴ and R ⁵ are H.

Residues of monosaccharide or oligosaccharide of R ⁷ are usually those that have a residue at the 1-position of the original end instead of 1-position or oligosaccharide monosaccharide. The constituent sugar of the monosaccharide and the oligosaccharide may be an amino sugar or a sugar acid or a derivative thereof. As the monosaccharide, D-glucosamine, D-galactosamine, D-mannosamine, D-galactose, D-darucose, D-mannose, D-glucuronic acid, L-iduronic acid and derivatives thereof are preferable. Consists of oligosaccharides in which the same monosaccharide is polymerized, and peronic acid selected from L-iduronic acid and D-glucuronic acid, and hexosamine selected from D-glucosamine and D-galactosamine. Glycosaminoglycans having a repeating structure of disaccharide units as a basic skeleton and derivatives thereof are preferred. That is, a disaccharide or glycosaminodalican in which the sugar residue at the reducing end has the structure of a monosaccharide described as the above general formula (1) can also be used in the production method of the present invention. The above-mentioned derivatives include those obtained by acetylating NH ₂ of the constituent sugars of monosaccharides or oligosaccharides and those obtained by sulfating OH.

In the present invention, an oligosaccharide is an oligosaccharide in the ordinary sense composed of two or more monosaccharides, and is usually 2 to 20 or more sugars or 2 to 20 sugars.

Substituents selected for R ² , R ³ , RR ⁵ and R ⁶ , including those for selecting substituents containing monosaccharide and oligosaccharide residues, are actually involved in the oxazoline formation reaction of the present invention. If the functional groups are not qualitatively involved and are presumed to be highly reactive during the oxazoline formation reaction of the present invention as in the ordinary synthesis reaction, it is necessary to prevent these from reacting. For example, a method may be considered in which the target substance is obtained by protecting with a protecting group used in a usual manner before subjecting to the above reaction of the present invention, and removing the protecting group after completion of the reaction. Therefore, in the acylamino sugar represented by the general formula (1), the group presumed to have high reactivity needs to be protected.

The protective group is not particularly limited as long as it does not hinder the reaction in the presence of the metal fluoride of the acylamino sugar represented by the general formula (1), but specific examples include an acetyl group, a benzoyl group, Methyl benzoyl group, bivaloyl group, levulinyl group, t-butyloxycarbonyl group, etc., lower alkyl group such as methyl group, ethyl group (usually about 1 to 5 carbon atoms), benzyl group, phenethyl group, etc. Alkylidene groups such as aralkyl groups and isopropylidene groups, and aralkylidene groups such as benzylidene groups, and aryl groups are preferably used. As the protective group, a lower alkyl group or an aralkyl group is preferably used. Usually, the protecting group is appropriately selected according to the type of the group to be protected.A plurality of types of groups may be protected by the same type of protecting group, or a plurality of the same type of group may be protected by a plurality of types. It may be protected by any kind of protecting group.

Preferred examples of the acylamino sugar used in the present invention include the following Examples 1 to 9.

Example 1) X: C 1, R 1: CH 3, R 2: 0 CO CH 3, R 3: H, R 4: H, R 5: ◦ CO CH 3, R 6: 0 CO CH generally a ₃ An acylamino sugar represented by the formula (1). Example 2) X: C 1, R 1: CH 3 R 2: OCOCHss R 3: H, R 4: 0 CO CH 3, R 5: H, R 6: 〇 CO CH ₃ a is general formula (1) Asilamino sugar indicated. Example 3) X: C 1, R 1: CH 3, R 2: 0C_〇_CH _{^{3, R 3: H, R}} 4: H, R 5:? 0- / - D- galacto Ichisu (0H is Asechiru group ), An acylamino sugar represented by the general formula (1), wherein R ⁶ : 0 C〇CH ₃ .

Example 4) X: C 1, R 1: CH 3, R 2: 0- D- glucuronic acid (OH is protected by Asechiru group, and those protected with C00H Gabe Njiru group), R ^3: H , R ⁴ : H, R ⁵ : OCOCH ₃ and R ⁶ : OCOCH3, an acylamino sugar represented by the general formula (1).

Example 5) X: C 1, R 1: CH 3, R 2: 〇 one D- glucuronic acid (0H are protected by Asechi group, and those protected with C00H Gabe Njiru group),: R ³ : H, R ⁴ : OCOCH ₃ , R ⁵ : H, ⁶ : 0 COCH ₃ , an acylamino sugar represented by the general formula (1).

Example 6) X: C 1, R 1: CH 3, R 2: 0- Fei _L- Izuron acid (0H is protected by Asechiru group, and those protected with C00H Gabe Njiru group), R ^3: ^{_{H, R 4: 0C0CH 3,}} R 5: H, R 6: § sheet Ruamino sugar represented by 0C0CH ₃ a is formula (1).

Example 7) X: C 1, R 1: CH 3, R 2: 〇_C0CH _{^{3, R 3: H, R}} 4: H, R 5: 0- 5- D - Darukosamin (0H and NH ₂ are both Asechiru group ), An acylamino sugar represented by the general formula (1), wherein R ⁶ : 0 CO CH ₃ .

Example 8) X: C 1, R 1: CH 3, R 2: OCOCH 3, R 3: H, R 4: H, R 5: 0- 5-N- Asechiru -D - Darukosamin (OH is at Asechiru group Protected), R ⁶ : ◦ An acylamino sugar represented by the general formula (1), which is CO CH ₃ .

Example 9) X: C 1, R ¹ : CH ₃ , R ² : 0 C 0 CH ₃ , R ³ : H, R ⁴ : H, R ⁵ : 0—Peronic acid residue on the reducing end side of heparin ( ? - D - Guruku port emissions Sanzanmotomata Wahi - L - Izuron 0H and NH ₂ in which the sugar residue (unsulfated having an acid residue) is protected by Asechi group, and, C00H Gabe Njiru group ), An acylamino sugar represented by the general formula (1), which is R ⁶ : 0C 0CH ₃ (having C 1 at the reducing end and having an amino group at the 2-position acetylated) Heparin that has a glucosamine residue, other non-sulfated hydroxyl groups and amino groups are acetylated, and carboxyl groups are benzylated)

In addition, the acylamino sugar shown in Example 1 above protects 0H at positions 3, 4, and 6 of N-acetyl-D-glucosamine, which is a constituent sugar of chitin, with an acetyl group, and converts 〇H at position 1 to a chlorine atom. It has been replaced. The acylamino sugars shown in Example 4 above were synthesized from 0H (positions 4 and 6 of hexosamine, positions 2, 3 and 4 of peronic acid) in the disaccharide unit composed of hyaluronic acid peronic acid and hexosamine. ) Protected with acetyl group Then, the carboxyl group of the peronic acid residue is protected with a benzyl group, and the OH at the 1-position of the hexosamine residue is replaced with a chlorine atom. The acylamino sugar shown in Example 5 above has the 〇H (positions 4 and 6 of hexosamine and the positions 2, 3 and 4 of hexonic acid) in the disaccharide unit composed of peronic acid and hexosamine of chondroitin. It is protected with a cetyl group, the carboxyl group of a peronic acid residue is protected with a benzyl group, and the OH at position 1 of the hexosamine residue is replaced with a chlorine atom. The acylaminosugar shown in Example 6 above is the OH in the disaccharide unit composed of peronic acid and hexosamine of dermatan sulfate (positions 4 and 6 of hexosamine, positions 2, 3, and 4 of peronic acid) Is protected with an acetyl group, the carboxyl group of the peronic acid residue is protected with a benzyl group, and the OH at the 1-position of the hexosamine residue is substituted with a chlorine atom.

The metal fluoride used in the production method of the present invention is not particularly limited, but is preferably an alkali metal fluoride, more preferably sodium fluoride, lithium fluoride, rubidium fluoride, or cesium fluoride. And the like. In the case of production on an industrial scale, sodium fluoride fluoride is particularly preferred in consideration of handling as a reagent and price.

In addition, as generally used, metal fluoride may be used by being held on an inorganic solid carrier. Examples of the inorganic solid carrier include alumina, silica gel, magnesium oxide, molecular sieves (for example, Linde 4A (trade name)), clay (for example, montmorillonite), and diatomaceous earth (for example, celite (trade name)). And alumina is particularly preferred.

The conditions for the reaction between the acylamino bran represented by the general formula (1) and the metal fluoride are as long as the acylamino sugar used in the present invention reacts with the metal fluoride to produce a bicyclic sugar oxazoline derivative. There is no particular limitation, and those skilled in the art can appropriately set the parameters.

The solvent for the reaction is not particularly limited as long as it does not react with the used acylamino sugar and metal fluoride and can dissolve the used amino amino sugar, but it is not limited to acetonitril (CH ₃ CN), dimethyl sulfoxide (DMSO), dimethylform Amide (DMF), tetrahydrofuran, xylene and the like are preferred, CH ₃ CN, DMSO, and DMF are more preferred, and CH ₃ CN is particularly preferred. The reaction conditions such as the amount of the metal fluoride used, the reaction temperature and the reaction time are appropriately set depending on the amount of the acylamino sugar used and the like. The molar ratio of the acylamino sugar to the metal fluoride is preferably 1: The reaction temperature is preferably from room temperature to the boiling point of the solvent, more preferably from 30 to 60 ° C. When the reaction is carried out at the boiling point of the solvent used, a reflux condenser or the like can be used. The reaction time is preferably 0.5 hours to 3 days.

When setting the reaction conditions, it is preferable to confirm the completion of the reaction. A specific example of a method for confirming the completion of the reaction is thin-layer chromatography.

The reaction between the acylamino sugar represented by the general formula (1) and the metal fluoride is preferably performed in an atmosphere such as argon or nitrogen in order to avoid a reaction with water. As a method for purifying the target substance, a soxazoline derivative, from the reaction-terminated liquid, it is possible to appropriately select and use a conventional purification method.For example, insoluble substances are removed from the reaction-terminated liquid, and then After removing the water-soluble substance dissolved in the reaction-completed solution by a liquid separation operation, purification by silica gel chromatography, recrystallization or the like can be mentioned.

The method of removing the insoluble matter from the reaction-terminated liquid may be any method as long as the insoluble matter (solid) and the solution (liquid) are separated, and a commonly used filtration method using a glass filter, ceramic, or the like can be used.

In the method for producing the sugar oxazoline derivative represented by the general formula (3), the sugar oxazoline derivative represented by the general formula (2) is obtained by the above method, and at least a part of the protecting group of the obtained sugar oxazoline derivative is removed. It is characterized by being removed. According to this method, a saccharide-containing oxazoline derivative represented by the general formula (3) can be obtained in which at least a part of the protecting group of the saccharide-containing oxazoline derivative is removed. Removal of the protecting group can be performed according to a conventional method.

The sugar oxazoline derivative obtained by the production method of the present invention is considered to be used as a substrate for polymer synthesis utilizing ring-opening polymerization of an oxazoline ring. In particular, as the acylamino sugar of the production method of the present invention, R ^{2 to} R ^A sugar oxazoline derivative obtained by using a compound in which at least one of ⁶ is a monosaccharide or an oligosaccharide (a disaccharide or glycosaminoglycan having a specific structure at the reducing end) utilizes an enzyme-catalyzed polyaddition reaction It is thought that it can be used as a synthetic substrate for glycosaminoglycans. For example, a bicyclic sugar oxazoline derivative having an oxazoline ring at the reducing end of a lactosamine chain containing at least one basic skeleton composed of lactosamine disaccharide is obtained by the production method of the present invention, and the obtained derivative is used as a substrate. Lactosamine chain can be extended by allowing keratanase to act. Similarly, using a bicyclic sugar oxazoline derivative having an oxazoline ring at the reducing end of a hyaluronic acid chain containing at least one basic skeleton composed of a hyaluronic acid disaccharide obtained by the production method of the present invention as a substrate, Hyaluronic acid chains can be extended by the action of the enzyme. In addition, chitin or chitosan is obtained by reacting chitinase with dalcosamine having an oxazoline ring obtained by the production method of the present invention or a disaccharide having N-acetylglucosamine or dalcosamine bound to its non-reducing terminal as a substrate. It seems that it is also possible. Example '

Hereinafter, the present invention will be described more specifically with reference to examples. Example 1 Synthesis of bicyclic oxazoline using metal fluoride

Under the respective reaction conditions described in Table 1 below, synthesis was performed according to the following procedure.

Under argon atmosphere, add 1.8 g (5 mmol) of N-acetyl-3,4,6-tri-0-acetyl-hi-glucosaminyl in acetonitrile solution in potassium fluoride or cesium fluoride in 75 ml or 1.8 g (5 mmol) of a solution of N-acetyl-3,4,6-tri-0-acetyl-hy-darcosaminyl chloride in dimethylformamide (75 ml) was added, and the mixture was reacted under reflux. After confirming the completion of the reaction by thin layer chromatography, the mixture was filtered through a glass filter G4. The filtrate was concentrated in an evaporator, diluted with an excess amount of black-mouthed form, and separated with a saturated solution of sodium hydrogencarbonate and then with cold water until the pH of the solution became neutral. After the collected organic layer was dried over anhydrous sodium sulfate, sodium sulfate was removed by filtration, and the filtrate was concentrated by evaporation. The obtained residue was subjected to silica gel flash column chromatography (gel: silica gel 60, manufactured by Merck, particle size 0.040-0.063, developing solvent: hexane / ethyl acetate = 2/3 (volume Ratio)), the developed solution was further concentrated by evaporation and dried under reduced pressure, and yellow methyl syrup-like 2-methyl (3,4,6-tri-〇-acetyl-1,2-dexoxy) was obtained. -H-Darcovirano) [2, 1-d] -2-oxazoline was obtained. The yield is shown in Table 1.

Metal WhitelU ¹ solvent Reaction temperature Reaction time Yield

Fluoride (° C) (hour) (%)

KF 1 CH ₃ CN 60 24 71. 4

5 CHaCN 60 78 81.3

10 CHaCN 60 88 78. 7

10 DMF 60 24 66.2

C s F 1 CHsCN main, ½) 1 24 9.5

5 CHsCN room ¾m 24 10.0

10 CHaCN 60 1 34. 7

10 DMF 24 18.5

* 1: N-acetyl-3,4,6-tri-O-acetyl-chloride-darcosaminyl chloride

Equivalent (10 equivalents of potassium fluoride is 2.9 g,

10 equivalents of cesium fluoride are 7.6 g). The obtained product (2-methyl (3,4,6-tri-0-acetyl-1,2-dideoxy α-glucovirano) [2 , 1-d] -12-oxazoline) was as follows.

^{J H NMR (250 MHz, CDC1} 3): 5.95 (1H, d, Ji, 2 = 7.34 Hz, Anomapuroton), 2.0-2.1 (12H, in, methyl protons of the methyl protons and Okisazorin acetate).

^{1 3 C NMR (62.9 MHz,} CDCls): 99.4 (C- 1), 20.7- 20.9 (3C, methyl acetate

C), 13.9 (methyl C of oxazoline), 169.2-170.6 (30, carbonyl of acetyl

C), 166.7 (C on the oxazoline ring: 0-C2N) Industrial applicability

According to the present invention, an inexpensive and simpler production method of a sugar oxazoline derivative that can be mass-produced is provided.

Claims

The scope of the claims

1. A process for producing a sugar oxazoline derivative represented by the following general formula (2), which comprises reacting an acylamino sugar represented by the following general formula (1) with a metal fluoride.

Wherein X is selected from F, Cl, Br and I;

R ¹ is selected from H and (CH ₂ ) _n _CH ₃ (n = 0-5);

R ² , R ³ , R ⁴ ,: ⁵ and: R ⁶ are each independently H, N ₃ , OH protected by a protecting group, NH ₂ protected by a protecting group, and Y—R ⁷ (Y is 0 or NH or S, R ⁷ is a residue of a monosaccharide or oligosaccharide, and when OH, NH ₂ or COOH is present, a monosaccharide whose group is protected by a protecting group. Or a residue of an oligosaccharide). However, at least one of R ² and R ³ and at least one of R ⁴ and R ⁵ are H. )

(Wherein, RR \ R ³ , R ⁴ , R ⁵ and R ⁶ are the same as described above.)

2. The method according to claim 1, wherein the metal fluoride is an alkali metal fluoride.

3. reacting the acylamino sugar represented by the following general formula (1) with a metal fluoride, and then removing at least a part of the protecting group of the obtained sugar oxazoline derivative; 3) A method for producing a sugar oxazoline derivative represented by the formula:

(Where X is selected from F, Cl, Br and I;

R ¹ is selected from H and (CH ₂ ) _n — CH ₃ (n = 0-5);

RRR ⁵ and R ⁶ are each independently H, N ₃ , OH protected by a protecting group, NH ₂ protected by a protecting group, and Y—R ⁷ (Y is 0 or NH or S R ⁷ is a residue of a monosaccharide or an oligosaccharide, and when OH, NH ₂ or CO OH is present, these groups are the residues of a monosaccharide or an oligosaccharide protected by a protecting group. Is selected from However, at least one of R ² and R ³ and at least one of R ⁴ and R ⁵ are H. )

(Wherein, R ¹ is as defined above,

R ² , R ³ , R ⁴ , R ⁵ and R ^{6 each} independently represent H, N ₃ , OH optionally protected by a protecting group, NH ₂ optionally protected by a protecting group, And Y—R ⁷ (Y is 0 or NH or S, and R ⁷ is a residue of a monosaccharide or an oligosaccharide, and when OH, NH ₂ or CO OH is present, these groups are protecting groups. Or a monosaccharide or oligosaccharide residue which may be protected by However, at least one of R ² and R ³ and at least one of R ⁴ and R ⁵ are H. )

4. The method according to claim 3, wherein the metal fluoride is an alkali metal fluoride.